Many diagnostic immunoassays are known which generally employ the specific binding characteristics that exist between an analyte or protein with a specific antibody tagged with some traceable substituent. One problem which has long been associated with this method is how to remove excess antibody from the biological fluid being tested for analyte in a manner whereby the analyte concentrations can be accurately measured.
Various attempts to remove excess antibody include U.S. Pat. No. 4,298,682 which discloses the absorption of unreacted antibody on a solid phase consisting of a polyacrylamide gel sensitized to the specific antibody.
U.S. Pat. No. 4,551,425 to Freytag et al. discloses another method to remove excess antibody in an immunoassay for digoxin. Here, excess labeled antibody is removed by passing it through an affinity column that has ouabain, an analog of digoxin, immobilized on the solid phase chromatography matrix. The excess antibody is absorbed by the ouabain. The chromatography eluant is then examined for the labeled antibody-analyte complex.
While the above can be effective, they are all subject to improvement. In the case of the above-described solid phase separation techniques, the compound immobilized on the solid phase must exhibit sufficient affinity for the labeled antibody to be removed. Ideally, the immobilized compound is irreversibly bound to the solid phase. However, disassociation of the immobilized compound from the solid phase due to leaching can not be avoided. Changes in temperature and microbial contamination are unavoidable and may accelerate this process. This freed material, because of its affinity for antibody, will recognize and complex with labeled antibody producing undesired background interference and reduced assay sensitivity. Disassociation of immobilized compound from the solid phase material is a major contributor to reduced shelf life of the solid phase/immobilized compound complex.
Known compounds useful for immobilization on solid phase material in an immunoassay for digoxin include digoxin and ouabain. These compounds exhibit relatively high affinity for labeled anti digoxin antibody. However, these same compounds when freed from the solid phase are recognized by the anti-digoxin antibodies; complexing with the antibodies and producing undesired background interference. Loss of relatively small amounts of these compounds adversely effect assay performance. During synthesis or storage of digoxin or ouabain solid phases, disassociation from the solid phase complex will reduce the useful life of the digoxin or ouabain solid phase complex.
Accordingly, there is a need for an improved diagnostic immunoassay by solid phase separation for digoxin which provides a compound exhibiting sufficient antibody binding characteristics when immobilized on the solid phase and which also exhibits low recognition for digoxin antibody when freed from the solid phase material.